Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin...

Hyphal Development in Candida albicans Requires TwoTemporally Linked Changes in Promoter Chromatin forInitiation and MaintenanceYang Lu, Chang Su, Allen Wang, Haoping Liu*

Department of Biological Chemistry, University of California, Irvine, California, United States of America

Abstract

Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungalinfections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungalpathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast andhyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphaldevelopment, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here weshow that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes.Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis viaactivation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase underreduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module,leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specificgenes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. Thesequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signalingprovides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments inpathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a uniquemechanism for integrating multiple signals during development and cell fate specification.

Citation: Lu Y, Su C, Wang A, Liu H (2011) Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin forInitiation and Maintenance. PLoS Biol 9(7): e1001105. doi:10.1371/journal.pbio.1001105

Academic Editor: Joseph Heitman, Duke University Medical Center, United States of America

Received December 7, 2010; Accepted May 31, 2011; Published July 19, 2011

Copyright: � 2011 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the US National Institutes of Health grant R01GM/AI55155 to HL. HL was also supported in part by the NIH NationalInstitute of General Medical Sciences Center grant GM76516. The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

Abbreviations: ChIP, chromatin immunoprecipitation; HAT, histone acetyltransferase; HDAC, histone deacetylase; PKA, protein kinase A; TOR, target ofrapamycin

* E-mail: [email protected]

Introduction

Many organisms or cells are able to alter their phenotype or

developmental fate in response to changes in their environment, a

phenomenon referred to as plasticity. Plasticity is seen in a broad

range of biological systems from embryo development to memory

formation where long-lasting effects on gene expression outlive an

initial transient signal. The dynamic process of cell fate

specification is determined by a network of regulatory genes.

The architecture of the network defines the temporal order of

specification events. Therefore, understanding temporal dynamic

regulation of gene expression in response to extracellular signals is

critical for our comprehension of cell fate specification and

plasticity.

Morphological plasticity is a defining feature of Candida albicans,

a major opportunistic fungal pathogen of humans [1]. C. albicans

resides as harmless commensal flora in the gastrointestinal tract

and mucosal membranes of healthy individuals, but when the host

immune system is suppressed, the fungus can disseminate and

cause systemic infections. Unlike many other pathogenic fungi that

exist primarily in either yeast or hyphal forms and infect a specific

organ, C. albicans is able to undergo reversible morphological

changes between yeast, pseudohyphal, and hyphal forms of growth

in response to environmental cues and can successfully infect many

different anatomical sites of the human host. Its morphological

plasticity is the most important virulence attribute of C. albicans [2].

Hyphae have invasive properties that can promote tissue

penetration and escape from immune cells, whereas yeast cells

are suited for dissemination in the bloodstream. Several of the

genes that are specifically expressed in hyphae encode virulence

factors. For example, HWP1, ALS3, and RBT5 encode cell wall

proteins that are important for adhesion to host cells and iron

acquisition from the host [3–6].

Several signal transduction pathways are involved in the

regulation of hyphal development. Among them, the cAMP-

dependent protein kinase A (PKA) pathway plays an essential role

in hyphal morphogenesis and virulence [7]. The adenylate cyclase

Cyr1 and its associated protein are indispensable for hyphal

growth under all conditions [8–10]. Tpk1 and Tpk2 are catalytic

subunits of PKA; each plays distinct functions in hyphal

development [11–13]. Efg1 and Flo8, two transcription regulators

essential for hyphal development and virulence [14–16], are

PLoS Biology | www.plosbiology.org 1 July 2011 | Volume 9 | Issue 7 | e1001105

implicated to function downstream of the cAMP-PKA pathway

[17,18]. The hyphal transcriptional program is repressed by Tup1

through sequence-specific DNA-binding proteins [19–27], of

which Nrg1 plays a major role. nrg1 mutant cells are constitutively

filamentous under all conditions, similar to tup1 cells [25,26].

Ectopic expression of NRG1 inhibits hyphal filamentation in all

growth conditions [28,29]. Although molecular genetic analyses

have identified a number of key transcriptional regulators of

hyphal morphogenesis, our understanding of the transcriptional

regulation that governs the yeast-to-hypha transition remains

rudimentary.

The yeast-to-hypha transition can be induced by a wide range

of media and environmental conditions in vitro [1]. Serum in

combination with a rise in temperature to 37uC gives the most

robust induction of hyphae. Simple inoculation of stationary cells

into fresh medium at 37uC is also a powerful but transient inducer

of hyphae [30]. Several induction signals are transmitted through

Cyr1, including CO2/HCO32 and peptidoglycan found in serum,

as well as a rise in temperature [31–33]. Cyr1 is also regulated by

Ras1, Ras2, and Gpa2 in response to nutrients [34–38]. Farnesol,

a quorum-sensing molecule secreted to the medium by C. albicans

cells as a cell density signal [39], exerts its inhibitory effects on

germ-tube formation through Ras1-Cyr1 [40]. However, many

widely used hyphal-inducing media are poor in nitrogen and

carbon sources that are not favorable for the activation of the

cAMP-PKA pathway, raising the question of how C. albicans can

undergo hyphal development in both rich and poor media. The

target of rapamycin (TOR) protein kinase pathway is another

major nutrient-sensing pathway conserved in C. albicans [41].

Rapamycin can both inhibit hyphal formation on solid medium

and promote cell aggregation in liquid medium in C. albicans

[41,42], but it does not induce yeast-to-hyphal development.

Transcriptional activation or repression in response to external

stimuli is often mediated through dynamic changes in promoter

chromatin structure. This regulation occurs at many different

levels, including posttranslational modification of histones [43],

chromatin remodeling [44], incorporation of histone variants [45],

and cotranscriptional chromatin disassembly and assembly

[46,47]. All of these dynamic processes work in concert to

establish or alter the local properties of chromatin, although the

relative importance and order of these processes vary at each

individual promoter. Histone acetyltransferases (HATs) and

deacetylases (HDACs) play important roles in regulating chroma-

tin structure and transcription [48]. Histone acetylation is a

dynamic reversible process, and the balance of histone acetylation

is important for proper cellular functions [49]. We have previously

shown a dynamic nucleosomal H4 acetylation at the promoters of

hypha-specific genes during hyphal induction [50]. H4 histone

acetyltransferase complex NuA4 is recruited to promoters in both

yeast and hyphal forms and is required for the induction of hyphal

genes [50]. Here we show that the dynamic increase and decrease

in H4 acetylation at the promoters during hyphal induction

correlates with the sequential dissociation and association of two

different HDACs, Rpd3 and Hda1, respectively. Concomitant

with the dissociation of Rpd3 upon hyphal initiation, Nrg1 protein

disappears from cells in response to the activation of the cAMP-

PKA pathway, a step essential for hyphal initiation. Although

Nrg1 protein returns after hyphal initiation, promoter recruitment

of Hda1 under reduced Tor1 signaling leads to deacetylation and

eviction of the NuA4 HAT module, nucleosome reassembly, and

inhibition of Nrg1 access to the promoters of hypha-specific genes,

a step essential for sustained hyphal development. Importantly,

disappearance of Nrg1 during hyphal initiation is a prerequisite for

promoter recruitment of Hda1. The sequential regulation of

hyphal development by the activation of the cAMP-PKA pathway

and reduced Tor1 signaling provides a molecular mechanism for

the plasticity of dimorphism and how C. albicans adapts to the

varied host environments in pathogenesis.

Results

Yeast-to-Hyphal Development Involves Two TemporallyRelated Phases of Removing Nrg1 Inhibition

The transcription factor Nrg1 plays an essential role in

repressing hyphal development [22,25,26,51]. The significance

of Nrg1 is underscored by recent phenotypic profiling of 143

transcriptional regulator knockout mutants, where only nrg1 and

tup1 mutants are filamentous under all conditions examined [52].

Relief of the transcriptional repression by Nrg1-Tup1 may be the

essential and regulated step for the activation of the hyphal

transcriptional program under all conditions. Indeed, NRG1

transcript level has been shown to be reduced in hyphae

[22,25,26,53]. However, how Nrg1 is regulated at the protein

level and whether Nrg1 directly binds to the promoters of hypha-

specific genes is not clear. By chromatin immunoprecipitation

(ChIP) of C-terminal Myc-tagged Nrg1, we found that Nrg1 was at

the promoters of hypha-specific genes HWP1, ALS3, and ECE1

during yeast growth (Figure 1). We next analyzed the levels of

promoter-bound Nrg1 during yeast to hyphal morphological

development under four related conditions in YPD medium that

induce three distinctive developmental responses (Figure 1A). Cells

inoculated to YPD with or without serum at 30uC remain in the

yeast growth form. Cells inoculated into YPD at 37uC in the

absence of serum show normal germ tube formation, but are

unable to maintain hyphal development. Serum, in combination

with a shift in temperature to 37uC, induces robust and sustained

hyphal growth. By a time course ChIP, we found that, upon

hyphal induction, Nrg1 dissociated rapidly from the promoters

and remained at low levels during hyphal elongation in YPD with

serum (Figure 1B). In contrast, levels of promoter-associated Nrg1

Author Summary

Many organisms are able to change their phenotype inresponse to changes in the environment, a phenomenonreferred to as plasticity. Candida albicans, a majoropportunistic fungal pathogen of humans, can undergoreversible morphological changes between yeast (spheri-cal) and hyphal (filamentous) forms of growth in responseto environmental cues. This morphological plasticity isessential for its pathogenesis and survival in its hosts. Inthis study, we show that hyphal development is initiatedand maintained by two major nutrient-sensing cellulargrowth pathways that act by removing the inhibitionprovided by the transcriptional repressor Nrg1. Whileinitiation requires a rapid but temporary disappearance ofNrg1 via activation of the cAMP-dependent protein kinaseA pathway, maintenance requires the recruitment topromoters of the Hda1 histone deacetylase under condi-tions of reduced signaling by the target of rapamycin(TOR) kinase, leading to chromatin remodeling that blocksNrg1 access to the promoters of hypha-specific genes. Weobserved that recruitment of Hda1 to promoters happensonly during the time window when Nrg1 is absent. Suchtemporally linked regulation of promoter chromatin bydifferent signaling pathways provides a unique mechanismfor integrating multiple signals in the regulation of geneexpression and phenotypic plasticity during developmentand cell fate specification.

Plasticity of Dimorphism


Figure 1. Initiation and maintenance of hyphal development by removing Nrg1 inhibition. An overnight culture of wild-type cellscarrying Nrg1-Myc (HLY3922) were diluted 1:50 into the indicated medium at 30uC or 37uC in the presence or absence of 10% serum, and cells werecollected at 0 min, 15 min, 30 min, 1 h, 2 h, 3 h, and 5 h for cell morphology (A), ChIP (B), and Western blot (C) experiments. ChIP DNA werequantitated by qPCR with primers at the UAS regions of HWP1, ALS3, and ECE1, as described [50]. The ADE2 coding region was used as a control. TheNrg1-Myc enrichment is presented as a ratio of HWP1, ALS3, or ECE1 IP (bound/input) versus the control IP (bound/input). The 0 h values on HWP1were set to 1.00. The ChIP data show the average of three independent qPCR data with error bars representing the SEM. Western analysis was carriedout using a peroxidase-conjugated anti-c-myc (Roche Diagnostics) antibody to assess levels of Nrg1-myc, and with an anti-PSTAIRE (Cdc28, SantaCruz) antibody for loading control.doi:10.1371/journal.pbio.1001105.g001



recovered after the initial decrease under the condition of YPD

without serum, which correlated with the conversion to the yeast

growth phase around 3 h after the induction (Figure 1A and 1B).

Unexpectedly, Western analyses indicated that Nrg1 protein levels

decreased sharply at 15 and 30 min upon hyphal induction at 37uCcoinciding with germ tube formation. However, the Nrg1 level

recovered after 1 h of hyphal induction (Figure 1C). Serum was not

required for the disappearance of Nrg1, but was critical for

sustained hyphal development and excluding Nrg1 from promoters

during hyphal elongation (Figure 1). The Western analysis of cells

after 3 h of hyphal induction could not discriminate Nrg1 protein

from apical cells and subapical cells. It is possible that Nrg1 protein

was absent in the apical cell of each hypha and, therefore, allowed

sustained hyphal growth from the apical cells. We determined Nrg1

localization by immunofluorescence in hyphal cells after 5 h of

growth in YPD with serum at 37uC and found Nrg1 in the nucleus

of both apical and subapical cells (Figure S1), excluding this

possibility. Our data suggest that hyphal development involves two

phases of releasing Nrg1 inhibition: the first for initiation and the

second for maintenance. We suggest that initiation requires a

transient down-regulation of Nrg1 protein level, while maintenance

requires a regulation that prevents Nrg1 from binding at the

promoters of hypha-specific genes.

The cAMP-PKA Pathway Is Required for theDown-Regulation of Nrg1 for Hyphal Initiation

Several lines of evidence suggest that the rapid and temporary

disappearance of Nrg1 protein upon hyphal induction is regulated

by the cAMP-PKA pathway. First, we found that Nrg1

disappearance required the cAMP-PKA pathway as Nrg1 protein

level did not show an obvious reduction in cyr1 and tpk2 mutants

(Figure 2A). Similarly, no obvious change in Nrg1 level was

observed in an efg1 or flo8 mutant (Figure 2A). Second, the shift to

37uC during hyphal induction was critical for the observed Nrg1

disappearance (Figure 1C). This is consistent with the report that

Hsp90 regulates hyphal development via Cyr1 in response to a

shift in temperature [33]. Third, the major quorum-sensing

molecule farnesol, at a physiological concentration, completely

blocked the down-regulation of Nrg1 (Figure 2A). Farnesol is

reported to exert its inhibitory effects on hyphal initiation through

Cyr1 [39,40]. Consistent with the inhibitory effect of farnesol on

the down-regulation of Nrg1, different folds of dilution during

inoculation could lead to different durations of Nrg1 down-

regulation (Figure 1C versus Figure 2A). We also observed that

hyphal initiation correlated with Nrg1 disappearance in all liquid

media, including nutrient-poor media (Figure 2B). However,

hyphal initiation in a poor medium required inoculation of cells

from a saturated overnight culture. This result is consistent with

the report that release from quorum-sensing molecules, such as

farnesol, triggers the yeast-to-hypha transition at 37uC indepen-

dent of the inoculation media [30]. The timing of hyphal initiation

was slower in medium with mannitol than that with glucose

(Figure 2B), consistent with the activation of the cAMP pathway by

glucose in addition to the release of inhibition from farnesol.

Together, our observations suggest that hyphal initiation involves

a down-regulation of Nrg1 protein in response to the activation of

the cAMP-PKA pathway by a combination of temperature shift,

release from inhibition by farnesol, and nutrients in rich media.

The NRG1 transcript has been reported to be reduced during

hyphal induction in serum containing medium [22,25,26,53]. We

found that the decrease in NRG1 expression during hyphal growth

was dependent on the cAMP-PKA pathway, as Cyr1 (unpublished

data) or Tpk2 was required for the reduction in NRG1 expression

(Figure 2C), consistent with the observation that NRG1 transcript

level was not down-regulated in the efg1 mutant during hyphal

induction in serum [26]. The transcriptional down-regulation was

essential for the initiation of hyphal development, as ectopic

expression of NRG1 under the MAL2 promoter in wild-type cells

could not initiate hyphal development even under robust

induction conditions (Figure 2D). However, the decrease in

NRG1 transcript alone is not sufficient to provide the temporal

dynamic change in Nrg1 protein level during hyphal development.

We observed that, by promoter shut-down experiments, Nrg1 was

unstable when cells were inoculated into fresh medium at 30uCeither from an overnight culture (Figure 2E) or a logarithmic

growing culture (unpublished data). Adding farnesol inhibited

Nrg1 degradation, suggesting that release from farnesol inhibition

during inoculation is important in triggering the degradation of

Nrg1. Therefore, the rapid and temporary disappearance of Nrg1

protein is the combined result of a cAMP-PKA dependent down-

regulation of NRG1 expression and a burst of Nrg1 degradation

upon release from farnesol during inoculation. This temporary

removal of Nrg1 is essential for hyphal induction.

Promoter-Recruitment of Hda1 Is Required for SustainedHyphal Development by Limiting Promoter Access toNrg1

One possible mechanism for the reduced promoter access by

Nrg1 during hyphal elongation is a change in promoter

chromatin. C. albicans has one class II HDAC Hda1 that functions

as a repressor for phenotypic switching [54]. We found that cells

deleted of HDA1 were unable to maintain hyphal growth. They

were impaired in sustained hyphal development and transcription

of hypha-specific genes, but had no detectable defects during

initial germ-tube formation (Figures 3AB, S2A and Table S1A).

Furthermore, levels of promoter-bound Nrg1 increased dramat-

ically in the hda1 mutant after hyphal initiation, suggesting that

Hda1 is required to prevent Nrg1 binding to the promoters for

sustained hyphal growth (Figures 3C and S2B). These observations

of the hda1 mutant are similar to that of wild-type cells inoculated

into rich medium at 37uC without serum (Figure 1). To determine

whether Hda1 functions directly on the promoters of hypha-

specific genes, we performed a time course ChIP of Hda1. We

found that Hda1 was recruited to the promoters during hyphal

induction in a serum-dependent manner (Figures 3D and S2C).

We suggest that serum-induced Hda1 recruitment to the

promoters of hypha-specific genes leads to a change in promoter

chromatin that is no longer accessible to Nrg1, leading to sustained

hyphal development.

In contrast to Hda1, we found that the Rpd3 HDAC

preferentially associated with hypha-specific promoters in yeast

cells and dissociated rapidly from the promoters upon hyphal

induction, similar to the dynamic dissociation of Nrg1 (Figure S3).

The sequential dissociation and recruitment of two different

HDACs to the hypha-specific promoters likely account for the

dynamic change in nucleosomal H4 acetylation levels during

hyphal induction with the peak H4 acetylation at 30 min [50].

The Promoter-Associated Hda1 Deacetylates the Yng2Subunit of NuA4 Histone Acetyltransferase (HAT)module, Leading to the Reduction of Yng2 and NuA4HAT at the Promoters of Hypha-Specific Genes

How does promoter-associated Hda1 prevent Nrg1 from

binding to the promoters of hypha-specific genes during hyphal

elongation? One potential mechanism is chromatin remodeling.

We have previously shown nucleosome reassembly and a decrease

in H4 acetylation at the UAS regions of hypha-specific genes



Figure 2. Hyphal initiation requires cAMP-PKA dependent down-regulation of Nrg1 protein. (A) Components of the cAMP-PKA pathwayand release from farnesol inhibition are required for down-regulation of Nrg1 protein during hyphal initiation. Western analysis of Nrg1-Myc duringhyphal induction in wild-type, cyr1, tpk2, efg1, and flo8 mutants. Cells of wild type and mutants carrying Nrg1-Myc were diluted 1:100 into freshYPD+10% serum medium at 37uC with or without 30 mM farnesol as indicated. (B) Cell morphology and Western blot analysis of wild-type cellscarrying Nrg1-myc. Cells from overnight cultures of the indicated OD600 were diluted 1:100 into Lee’s media with either mannitol or glucose. Adding30 mM farnesol into Lee’s glucose medium blocked Nrg1 down-regulation and hyphal development. (C) Tpk2 is essential for transcriptional down-regulation of NRG1 during hyphal induction. NRG1 mRNA levels were determined by qRT-PCR in WT and tpk2/tpk2 strains over a time course ofhyphal induction (YPD+10% Serum). The signal obtained from ACT1 mRNA was used as a loading control for normalization. The data show theaverage of three independent qRT-PCR experiments with error bars representing the SEM. (D) Constitutive expression of Nrg1 blocks germ tubeformation. Cells of wild-type and nrg1/nrg1 carrying Nrg1-Myc under the MAL2 promoter were grown in YPMaltose medium at 30uC overnight andwere diluted at 1:20 into pre-warmed YPMaltose medium at 37uC. (E) Nrg1 protein stability by promoter-shutdown. Western or wild-type cellscarrying Nrg1-Myc under the MAL2 promoter inoculated from overnight culture into fresh YPD medium at 30uC with and without 30 mM farnesol.doi:10.1371/journal.pbio.1001105.g002



during hyphal elongation [50]. The decrease in H4 acetylation

could be a result of H4 deacetylation by promoter-associated

Hda1 or eviction of NuA4 from the promoters. S. cerevisiae Hda1 is

a histone deacetylase specific for H3 and H2B [55]. Therefore, H4

may not be a substrate of the C. albicans Hda1. Yng2, a subunit of

the NuA4 histone acetyltransferase HAT complex essential for

HAT activity, is acetylated at lysine 170 by NuA4 and

deacetylated by Rpd3 in S. cerevisiae [56]. Deacetylation of Yng2

leads to its degradation and eviction of chromatin-bound Yng2

with Esa1, the catalytic subunit of NuA4. In C. albicans, the NuA4

HAT complex is recruited to the promoters of hypha-specific

genes [50]. By immunoprecipitation with anti-acetylated-lysine

antibodies, we detected acetylated Yng2 in wild-type cells, but

observed a dramatic increase in the level of acetylated Yng2 in the

hda1 mutant (Figure 4A). This suggests that deacetylation of Yng2

in vivo depends on Hda1 activity. K175 was identified as a

candidate lysine residue for acetylation by sequence alignment

with S. cerevisiae Yng2. This was confirmed by the loss of

acetylation of Yng2 when we substituted K175 with arginine

(K175R), a mutation that blocks acetylation (Figure 4B). The loss

of acetylation of Yng2K175R also suggests that K175 is the major

acetylated lysine residue of Yng2 in C. albicans. The K175R

mutation led to a reduced level of Yng2, whereas substituting

K175 with glutamine (K175Q, a mutation mimicking constitutive

acetylation) did not cause a detectable change of protein

abundance relative to wild type (Figure 4C). Unlike the yng2

deletion mutant, neither yng2K175R nor yng2K175Q has any

detectable growth defect (unpublished data). We then examined

whether Yng2 acetylation affects hyphal development. Both wild-

type and yng2K175R cells developed long hyphae, whereas yng2K175Q

cells were defective in sustained hyphal development and

transcription (Figures 4D,E and S4A), a phenotype similar to that

of the hda1 mutant. Therefore, Yng2 deacetylation at K175 is

important for sustained hyphal transcription. Conversely, consti-

tutively acetylated Yng2 blocks hyphal filament extension.

If serum-induced Hda1 promoter association leads to Yng2

deacetylation and degradation, the level of promoter-associated

Yng2 is expected to decrease during hyphal induction. Indeed, a

time course ChIP of Yng2-Myc showed that Yng2 level decreased

after 30 min of hyphal induction (Figures 4F and S4B). The level

of promoter-associated Yng2K175R showed a similar decrease,

whereas the levels of Yng2-Myc in the hda1 mutant and

Yng2K175Q-Myc stayed unchanged (Figures 4F, 5C, and S4C).

The level of Esa1, the enzymatic subunit of NuA4, showed a

similar decrease at the promoters during hyphal induction

(unpublished data).

To determine whether the deacetylation of Yng2 is the major

function of Hda1 in hyphal development, we generated an hda1

Figure 3. Promoter recruitment of Hda1 is required for hyphal maintenance by inhibiting Nrg1 access to the promoters of hypha-specific genes. (A, B) Hda1 is required for maintenance of hyphal development as shown by morphology and expression levels of hypha-specificgenes during hyphal induction. Cells of wild-type (TS3.3+pBES116) and hda1/hda1 (HLY4032+pBES116) strains were diluted into YPD with 10% serumat 37uC. HWP1, ALS3, and ECE1 mRNA levels were determined by qRT-PCR. The signal obtained from ACT1 mRNA was used as a loading control fornormalization. (C) Nrg1-Myc binds the promoters of hypha-specific genes in hda1 cells during hyphal elongation in YPD with 10% serum. (D) Serum-dependent binding of Hda1-Myc to the hyphal promoters in wild-type cells during hyphal induction. Cells of wild-type strain containing Hda1-Myc(HLY4033) were diluted into pre-warmed YPD medium at 37uC in the presence or absence of 10% serum. ChIP DNA in (C, D) were quantitated byqPCR with primers at the UAS regions of hypha-specific genes as described in Figure 1. Data for ALS3 and ECE1 from (B, C, D) are shown in Figure S2.All data show the average of three independent qRT-PCR or qPCR experiments with error bars representing the SEM.doi:10.1371/journal.pbio.1001105.g003



Figure 4. The function of Hda1 in sustained hyphal transcription is mediated through Yng2 deacetylation (A) Yng2p is deacetylatedthrough an Hda1-dependent mechanism in vivo. Cells were grown at 30uC to OD600 0.8, then WCEs were collected, immunoprecipitated with anti-Ac-K, and probed with anti-Myc. (B) K175 is the major acetylated lysine residue of Yng2 in vivo. Substitution of K175 with arginine (K175R) diminishesacetylation of Yng2. (C) Effects of K175 substitutions on Yng2 stability. K175R mutation causes decreased protein abundance of Yng2, while K175Qcauses no detectable change. (D, E) Morphology and expression levels of hypha-specific genes in YNG2 (HLY4035), yng2K175R (HLY4036), and yng2K175Q

(HLY4037) cells during hyphal development in YPD with 10% serum at 37uC. HWP1, ALS3, and ECE1 mRNA levels were determined by qRT-PCR. Thesignal obtained from ACT1 mRNA was used as a loading control for normalization. (F) Dynamic dissociation of Myc-tagged Yng2 and yng2K175R butnot Yng2K175Q from promoters during hyphal development by ChIP with anti-Myc antibodies. (G) ChIP with anti-H3 and anti-acetylated H4 antibodies



yng2 double mutant and introduced the yng2K175R mutation into

the double mutant. The hda1 yng2K175R mutant behaved like

yng2K175R or wild-type cells with no defect in hyphal filament

extension and transcription (Figure 5A and 5B). Furthermore,

Hda1 is required for the reduction of promoter-associated Yng2,

and the yng2K175R mutant completely bypassed this requirement of

Hda1 (Figures 5C and S5). Therefore, the function of Hda1 in

sustained hyphal transcription is mediated completely through the

deacetylation of Yng2 at K175. In addition, the level of promoter-

associated Yng2K175R decreased in the hda1 mutant as did Yng2 in

wild-type cells during hyphal elongation (Figures 5C and S5),

suggesting that Hda1 deacetylation of Yng2 at the promoters led

to the eviction of Yng2 and Esa1 from the promoters of hypha-

specific genes.

To understand the effect of Yng2 K175 deacetylation on

promoter chromatin, we determined temporal changes in histone

levels and H4 acetylation at the UAS regions of hypha-specific

promoters during hyphal induction (Figures 4G and S4D).

Changes in H3 levels indicated rapid nucleosome disassembly

during hyphal initiation and nucleosome reassembly during

hyphal maintenance. The observed decrease in H3 occupancy

and transcriptional induction within 30 min of hyphal induction is

consistent with the notion that transcriptional activation correlates

with reduced nucleosome occupancy [57]. In contrast, nucleosome

reassembly during hyphal elongation seemed important for

sustained hyphal transcription, as yng2K175Q cells showed a specific

defect in nucleosome reassembly without any detectable impair-

ment in initial nucleosome disassembly. Our data suggest that

nucleosome reassembly during hyphal maintenance may function

in preventing Nrg1 from binding to the promoters.

Reduced TOR Signalling Led to Hda1 PromoterRecruitment and Sustained Hyphal Development

yng2K175Q cells exhibited normal hyphal initiation in several

hyphal growth media in addition to serum (Table S1B) and were

defective in sustained hyphal development in all media that can

support prolonged hyphal development (Figure 6A). This

suggested that the Hda1-mediated deacetylation of Yng2 was

Figure 5. yng2K175R mutation suppresses the deficiency of hda1/hda1 in sustained expression of hypha-specific genes. Morphology (A)and Northern analysis (B) of wild type and hda1/hda1 yng2/yng2 transformed with YNG2 or yng2K175R during hyphal induction as in Figure 3. Northernanalysis was carried out using 12.5 mg RNA and probes to the indicated filament-specific transcripts. The ACT1 transcript is included as a loadingcontrol. (C). Kinetics of Yng2-Myc and Yng2-Myc-K175R promoter binding in hda1/hda1 mutants by ChIP with anti-Myc. Data on ALS3 and ECE1 for (C)are in Figure S5. The ChIP data show the average of three independent qPCR experiments with error bars representing the SEM.doi:10.1371/journal.pbio.1001105.g005

show temporal dynamics in relative levels of H3 and H4 acetylation at the promoters of hypha-specific genes during hyphal induction in the YNG2(HLY4035) and yng2K175Q mutant (HLY4037) strains. Levels of H3 (relative H3 occupancy; bound/input) are normalized to the respective control DNAat the ADE2 coding sequence region (bound/input). The 0-h values in the wild-type strain are set to be 1.00. Acetylation levels normalized withrespect to H4 levels (H4 acetylation/H3 occupancy) are calculated by dividing the values for acetylated H4 with H3 occupancy values. Data on ALS3and ECE1 for (E, F, G) are in Figure S4. All data show the average of three independent qPCR experiments with error bars representing the SEM.doi:10.1371/journal.pbio.1001105.g004



required for hyphal maintenance in those media. In addition,

Hda1 was found to be associated with the promoters of hypha-

specific genes, whereas Nrg1 promoter-association was inhibited

after 6 h of growth in those conditions (Figure 6B). Interestingly,

those media tend to be nutrient-poor. One commonly used

synthetic nutrient-poor medium for hyphal growth is Lee’s

medium [58]. It contains glucose, ammonium sulfate, and many

amino acids, but lacks glutamine, a preferred nitrogen source. The

rapamycin-sensitive Tor1 kinase is a central regulator of cell

growth in response to nitrogen and amino acid availability in yeast

[59] and is conserved in C. albicans [41]. We hypothesized that a

nutrient-poor medium, such as Lee’s, is sensed by C. albicans cells

as nitrogen limiting, and therefore leads to reduced Tor1 activity.

If so, treatment of C. abicans cells with rapamycin in a rich medium

should mimic a nutrient-poor medium for sustained hyphal

development. As predicted, treatment of C. albicans cells with a

sublethal concentration of rapamycin after hyphal initiation at

37uC led to sustained hyphal filamentation in rich medium

(Figure 7A). A higher concentration of rapamycin, however,

slowed cell growth and hyphal elongation (unpublished data),

consistent with the finding that rapamycin inhibits filamentation

on nutrient-poor media [41]. Sustained hyphal filamentation was

also observed at 30uC with sublethal levels of rapamycin after cells

were first grown at 37uC for hyphal initiation (Figure 7A). The

effect of rapamycin on hyphal maintenance was only observed

after hyphal initiation. Cells treated with rapamycin at 30uC did

not form hyphae (Figure 7B). We further showed that the effect of

Tor1 on hyphal maintenance was mediated through the promoter-

recruitment of Hda1, as yng2K175Q cells were defective in sustained

hyphal filamentation in rapamycin (Figure 7C). In addition, Hda1

was recruited to the promoters of hypha-specific genes in cells

treated with rapamycin in rich medium, whereas Nrg1 was

excluded from the promoters (Figure 7D). The phenotypes of the

yng2K175Q cells and patterns of promoter association for Hda1 and

Nrg1 are similar between poor medium and rapamycin treatment

(Figures 6 and 7). Furthermore, a mutant with a hyperactive TOR

Figure 6. yng2K175Q mutant is defective in hyphal elongation in nutrient-poor media. (A) Cell morphology of wild-type (HLY4035) andyng2K175Q (HLY4037) cells grown at 37uC in YEP+GlcNAc, SCAA, M199 pH 8.0, or Lee’s with mannitol for indicated times. (B) ChIP with anti-Mycantibodies in wild-type cells carrying Hda1-Myc (HLY4033) or Nrg1-Myc (HLY3922). qPCR was used to quantitate ChIP DNA with primers at the UASregion of HWP1 under the indicated conditions. The ChIP data show the average of three independent qPCR experiments with error barsrepresenting the SEM.doi:10.1371/journal.pbio.1001105.g006



pathway in C. albicans has recently been shown to be unable to

form hyphae in a nutrient-poor medium, and rapamycin can

rescue the filamentation defect [60]. Therefore, maintenance of

hyphal development requires reduced Tor1 signaling in response

to nutrient-poor media and growth conditions. The reduced Tor1

signaling leads to the recruitment of Hda1 to the promoters of

Figure 7. Reduced Tor1 signaling allows sustained hyphal elongation through promoter recruitment of Hda1. (A) Treatment ofC. albicans cells with rapamycin after hyphal initiation at 37uC led to sustained hyphal filamentation in rich medium. Wild-type (HLY4035) cells weregrown in YPD medium at 30uC overnight and were diluted at 1:250 into pre-warmed YPD medium at 37uC or grown at 37uC for 1 h and thentransferred to 30uC. 5 nM rapamycin was added after 1 h to half of the samples. Cells were collected at 0 h, 1 h, 2 h, 4 h, and 8 h for cell morphology.(B) Wild-type cells grown in YPD at 30uC with and without 5 nM rapamycin for the indicated hours. (C) yng2K175Q (HLY4037) cells were grown in YPDat 37uC for 8 h. 5 nM rapamycin was added after 1 h to half of the samples. (D) Nrg1 protein levels and Nrg1-myc or Hda1-myc promoter associationduring hyphal growth in YPD at 37uC with and without 10 nM rapamycin. ChIP DNA were quantitated by qPCR with primers at the UAS region ofHWP1. The ChIP data show the average of three independent qPCR experiments with error bars representing the SEM.doi:10.1371/journal.pbio.1001105.g007



hypha-specific genes for promoter remodeling that prevents Nrg1

from binding.

Temporal Connection between Hyphal Initiation andMaintenance

To uncover potential molecular connections between the

initiation phase and elongation phase of hyphal development, we

asked whether serum could induce the promoter recruitment of

Hda1 without the initiation phase. We found that Hda1 did not

bind the promoters of hypha-specific genes when cells were grown

at 30uC in the presence of serum (Figure 8A). Therefore, serum is

not sufficient to induce the promoter recruitment of Hda1 without

the initiation step. We further showed that raising the temperature

to 37uC during inoculation was a prerequisite for subsequent

recruitment of Hda1 to the promoters (Figure 8A). Since Nrg1 was

temporally cleared when cells were inoculated into fresh medium

at 37uC, a requirement for hyphal initiation, we then examined

whether serum could induce promoter recruitment of Hda1

independent of temperature in the nrg1 deletion mutant by a time

course ChIP experiment. Hda1 was found to bind the HWP1

promoter in the presence of serum in nrg1 mutants in either 25uCor 37uC (Figure 8B). This result suggests that Nrg1 removal is

required and sufficient for serum-induced Hda1 promoter

recruitment for the sustained hyphal transcriptional program.

This also predicts that the sustained hyphal transcriptional

program could only be established within the time window of

reduced Nrg1 when cells had just been inoculated into fresh media

at 37uC. Indeed, addition of serum after 2 h of hyphal induction at

37uC in YPD could not sustain hyphal development (Figure 8C).

Similar to serum, rapamycin could sustain hyphal growth only

when added during hyphal initiation. Adding rapamycin after 2 h

of hyphal induction showed no effect on hyphal maintenance

Figure 8. The sustained hyphal growth program could only be established during the time window of reduced Nrg1. (A) Serum is notsufficient to induce the promoter recruitment of Hda1 without hyphal initiation at 37uC. ChIP with anti-Myc antibodies in wild-type cells carryingHda1-Myc in YPD with 10% serum under indicated conditions. Primers at the UAS region of HWP1 were used. (B) Nrg1 removal is required andsufficient for serum-induced Hda1 promoter recruitment. The ChIP time course was carried out with anti-Myc antibodies in nrg1/nrg1 cells carryingHda1-Myc under the indicated conditions. Cells were collected at 0 min, 15 min, 30 min, 1 h, 2 h, and 3 h. The ChIP data show the average of threeindependent qPCR experiments with error bars representing the SEM. (C) Serum and rapamycin are unable to sustain hyphal development when theyare added to YPD medium 2 h after inoculation at 37uC. Cells from overnight cultures in YPD at 30uC were diluted at 1:250 into pre-warmed YPDmedium at 37uC; 10% serum or 5 nM rapamycin were added at 1 h, 2 h, or 3 h after inoculation, respectively. The total incubation time is 5 h forserum and 8 h for rapamycin experiments.doi:10.1371/journal.pbio.1001105.g008



(Figure 8C). Together, our data show a clear temporal connection

between hyphal initiation and maintenance: Hda1 can be

recruited to the promoters of the hypha-specific genes only when

Nrg1 level is low. The time period of reduced Nrg1 during hyphal

initiation can be viewed as a window of opportunity for

establishing the hyphal transcription program. The strength and

duration of the cAMP signal in each cell is expected to affect the

commitment to the hyphal transcriptional program; once

committed, the cAMP-PKA pathway activation is no longer

needed. This is consistent with the observation that farnesol only

inhibits germ-tube formation and has no effect on hyphal

elongation [39]. The duration of hyphal development is

determined by nutrient conditions through the TOR pathway.

Discussion

This study shows that the yeast to hyphae morphogenetic switch

consists of two temporally related phases of regulation of the

promoter chromatin of hypha-specific genes: the first for initiation

and the second for maintenance of hyphal development (Figure 9).

Initiation requires the cAMP-PKA pathway and maintenance

requires reduced Tor1 signaling. A spike of cAMP-PKA activation

[10] and release from the inhibition of quorum sensing molecules,

such as farnesol, initiate hyphal development by transiently

clearing Nrg1 protein. The duration of hyphal development is

under the regulation of Tor1 by controlling promoter access to

Nrg1 through promoter recruitment of Hda1. Therefore, hyphal

maintenance requires active sensing of growth environments.

Hyphal cells convert to yeast when nutrients are replete. This

provides an underlying mechanism for the plasticity of dimor-

phism. Importantly, the sustained transcriptional program, as

measured by promoter recruitment of Hda1, can only be

established during the time period when Nrg1 is absent. This

provides a temporal link at the molecular level between the two

phases of hyphal development. This is the first example of a

temporal integration of two major nutrient-sensing cell growth

pathways in development. Temporal restriction in development of

multicellular organisms is common. For example, nutritional

control of the reproductive status in honeybees is restricted by a

critical ‘‘decision-making’’ period in larval development [61]. Our

study also provides a mechanism for how a transient signal, such as

a cAMP spike, can make a long-lasting impact in transcriptional

reprogramming during development. Such control of cellular

development by a burst of proliferating signal, followed with

sustained action of reduced TOR signaling has been observed for

memory T-cell differentiation [62]. The TOR level is also critical

for maintaining stem cell homeostasis [63–65]. Temporal

regulation of cell fate by different signaling pathways is likely

Figure 9. A schematic diagram depicting initiation and maintenance of hyphal development. (A) Hyphal development in C. albicansrequires two phases of sequential changes in the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporarydisappearance of Nrg1 in response to the activation of the cAMP-PKA pathway, while the duration of hyphal development is under the regulation ofthe TOR pathway by controlling promoter access to Nrg1 through recruitment of Hda1. Black lines represent active regulatory relationships; grey linesrepresent relationships that are inactive. Dashed circles represent degraded proteins. (B) Hda1 is recruited only during the time period when Nrg1 isabsent. This provides a mechanism for temporal integration of two major nutrient-sensing cell growth pathways in development.doi:10.1371/journal.pbio.1001105.g009



common in development of multicellular organisms. Coupling two

signaling pathways through the regulation of promoter chromatin

provides a unique mechanism.

Initiation of the hyphal transcriptional program requires

temporary removal of Nrg1 protein. The timing, duration, and

extent of the Nrg1 down-regulation are well correlated with timing

and efficiency of hyphal initiation and are sensitive to multiple

factors, including the state of inoculating cells (e.g., stationary

cells), fold of dilution at inoculation, media, and temperature. Of

those, a rise in temperature to 37uC and release from farnesol

inhibition are essential for the clearance of Nrg1 and hyphal

initiation. The cAMP-PKA pathway is required for the down-

regulation of Nrg1, consistent with the concept that Cyr1 is a hub

that integrates many of the hyphal inducing signals [31–33,40].

Efg1 and Flo8 are both required for the down-regulation of Nrg1,

but a TRA or TRE mutation at the sole conserved PKA site in

Efg1 (T206) does not reduce or enhance hyphal gene expression

(our unpublished data). If Efg1 activity is directly regulated by

PKA phosphorylation, the regulation is not essential or sufficient

for the induction of hypha-specific genes. Efg1 and Flo8 also

function directly on the promoters of hypha-specific genes [16,50],

and the tup1 mutant cannot bypass the requirement of Efg1 for

hyphal transcription [66]. Our finding that Nrg1 is negatively

regulated by the cAMP-PKA pathway and by farnesol is

intriguing. The nrg1 mutant produces 19-fold more farnesol than

the parent wild-type strain [67], and farnesol in turn impacts

activity of the Cyr1 pathway [40]. Together, these findings provide

a loop of three negative feedback regulations from farnesol to

Cyr1, to Nrg1, and then back to farnesol. Removal of farnesol

during inoculation will lead to activation of the cAMP-PKA

pathway and clearance of Nrg1 (Figure 9). Lack of Nrg1 in turn is

expected to lead to increased production of farnesol, and a new

balance among the components in the feedback loop will be

achieved. Another intriguing finding is that the sustained hyphal

transcriptional program can only be established during the time

window of low Nrg1. Commitment to the hyphal program is

therefore determined by the strength and duration of Nrg1 down-

regulation in each cell. If the duration is not long enough to

establish the Hda1-mediated chromatin remodeling, the cell will

be in the yeast growth phase. This explains the cell-to-cell

variation and experiment-to-experiment variation in hyphal

induction in a given culture, and why yeast and hyphal cells can

exist in the same culture. Our model explains a widely accepted

observation that the quality of the initial hyphal induction is

important for the fate of hyphal development. It also provides a

molecular mechanism for why farnesol only inhibits germ-tube

formation and has no effect on hyphal elongation [39].

Our finding of temporally coupled chromatin remodeling under

reduced Tor1 signaling for sustained hyphal transcription provides

a molecular mechanism for why nutrient-poor media are typical

media for sustained hyphal development. It has been puzzling how

C. albicans can respond to both rich and poor media and undergo

hyphal development. Rich medium with serum is the most robust

medium for hyphal induction, and this is consistent with the

requirement of the cAMP-PKA pathway for hyphal growth.

Hyphal induction in nutrient-poor media also requires an

activation of the cAMP-PKA pathway, mostly through the release

from farnesol inhibition. But sustained hyphal growth requires a

nutrient limitation. Considering the conserved functions of Tor1 in

nutrient sensing and growth regulation from yeast to human,

involvement of Tor1 in hyphal development has been studied and

implicated [41,60,68]. However, rapamycin cannot induce hyphal

development, making it hard to reconcile if the cAMP-PKA and

Tor1 pathways function in parallel. We find that rapamycin can

lead to the promoter recruitment of Hda1 only during hyphal

initiation in the absence of Nrg1. The temporal coupling of the

two pathways through the regulation of the promoter chromatin of

hypha-specific genes explains why rapamycin by itself cannot

induce hyphae. Since serum can sustain hyphal development in

YPD, we suspect it may contain certain components that are

inhibitory to the Tor1 pathway. The nuclear localization and the

activity of several nutrient-responsive transcription factors are

regulated by Tor1-mediated phosphorylation in yeast [69]. It is

possible that a Tor1-regulated transcription factor is necessary for

the recruitment of Hda1 to the promoters of hypha-specific genes.

Further studies are needed to address this. In addition to Hda1-

mediated chromatin remodeling for sustained hyphal transcrip-

tion, there is a built-in positive feedback on the hyphal

transcriptional program that is also important for sustained hyphal

development. The transcription factor Ume6, specifically ex-

pressed during hyphal development, controls the level and

duration of hypha-specific genes and is important for hyphal

elongation [53,70,71]. The strength of the cAMP and Tor1

signaling determines the level and duration of UME6 expression,

which in turn dictates the extent of hyphal morphogenesis. Hyphal

morphogenesis and cell chain formation is under the control of

another hypha-specific gene that encodes the G1 cyclin-related

protein Hgc1 [72–76].

Our study should provide insights into understanding C. albicans

pathogenesis. Hyphal initiation is tightly linked to release from

quorum-sensing molecules, a condition parallel to dispersion of C.

albicans cells from biofilms in human hosts, which is a major cause

of disseminated candidiasis [77]. Invasive candidiasis is the most

common invasive fungal infection among organ transplant

recipients [78]. Rapamycin is given to transplant patients,

especially renal transplant patients, to prevent organ rejection.

One speculation from our finding is that rapamycin, known for its

antifungal activity, may also facilitate hyphal development and

invasive infections. The existence of extensive hyphae in various

deep-seeded infection sites also suggests that those host environ-

ments may be perceived as stressful or starvation conditions by

Candida. Our findings provide molecular mechanisms for how C.

albicans adapts to varied host environments and develops from a

benign commensal into a disseminated invasive disease.

We have previously noticed the dynamic increase and decrease in

H4 acetylation concomitant with nucleosome disassembly and

reassembly at the promoters of hypha-specific genes during hyphal

induction [50]. The significance of the temporal dynamic regulation

in promoter chromatin is not clear. Here we find the sequential

dissociation of Rpd3 and association of Hda1 to the promoters is

concomitant with the dynamic changes in H4 acetylation.

Importantly, we show that promoter-associated Hda1 decreases

H4 acetylation level by deacetylating Yng2 and evicting Yng2 and

Esa1 of the NuA4 HAT module from the promoters, leading to

nucleosome reassembly, inhibition of Nrg1 binding, and sustained

hyphal transcription. This provides an example for Hda1 in gene

activation and NuA4 in gene repression. The function of NuA4 in

repression here is revealed by the use of yng2K175Q and yng2K175R

mutations. Temporal analysis of promoter chromatin also delineates

different functions of HDACs and NuA4 in transcription. It shows

that nucleosome reassembly does not necessarily correlate with

transcriptional repression; it can also be used for gene activation by

restricting access of repressors. In S. cerevisiae, Yng2 is deacetylated

by Rpd3 and a similar temporal dynamic H4 acetylation is observed

at DNA double-stranded breaks concomitant with the sequential

recruitment of NuA4 and Rpd3 [56]. Such temporal dynamic

regulation of chromatin is likely also used in transcription in S.

cerevisiae. Indeed Rpd3 is required for the expression of several



environment-responsive genes [79–82]. Active functions of HDACs

in gene expression are not limited to yeast. A recent genome-wide

mapping of HAT and HDAC binding sites in human T cells shows

that all HDACs analyzed are associated with active genes and

positively correlated with transcription, with some HDACs mainly

in the promoter regions and other HDACs in both the promoter

and gene body regions [83]. Inhibition of HDAC activity with

HDAC inhibitor treatment causes increases in acetylation in the

active genes, suggesting that the majority of HDACs function to

reset chromatin by removing acetylation at active genes [83]. The

temporal dynamic NuA4 regulation by HDACs shown in this study

provides an attractive mechanism likely used for temporal

integration of different signals in transcriptional reprogramming

during development.

Materials and Methods

Media and Growth ConditionsC. albicans strains were routinely grown at 30uC in YPD (2%

Bacto peptone, 2% dextrose, 1% yeast extract). Transformants

were selected on synthetic medium (2% dextrose, 0.17% Difco

yeast nitrogen base w/o ammonium sulfate, 0.5% ammonium

sulfate and auxotrophic supplements). M199 medium (Sigma-

Aldrich) was buffered at pH 8 using 150 mM HEPES. Hyphal

induction was performed as follows. Strains were grown overnight

in liquid YPD at 30uC, pelleted, washed twice in PBS, resuspended

in an equal volume of PBS, and diluted 1:100, unless otherwise

indicated, into YPD+10% serum (Sigma-Aldrich), Lee’s medium

[58] with modifications [5 g (NH4)2SO4, 2.5 g K2HPO4 Anhy-

drous, 5 g NaCl, 0.5 g alanine, 1.3 g leucine, 1 g lysine, 0.1 g

methionine, 0.072 g ornithine, 0.5 g proline, 0.5 g threonine,

10 ml 20 mg/ml MgSO4, 1 ml 2 mg/ml biotin, with pH adjusted

to 7.0, with either 1% glucose or mannitol, filled to 1 L with

water], YEP+2% N-acetyl-glucosamine (Sigma-Aldrich), SCAA

medium [15] with modifications [2% Casein Hydrolysate broth

(Sigma-Aldrich), 0.17% Difco yeast nitrogen base w/o ammonium

sulfate], Spider medium [1% mannitol, 1% nutrient broth, 0.2%

K2HPO4, pH 7.2 before autoclaving] [84], M199 pH 8 or

YPD+5 nM rapamycin. Cultures were grown at 37uC.

Plasmid and Strain ConstructionThe C. albicans strains used in this study are listed in Dataset S1.

Primer sequences are listed in Dataset S2. HDho15 and RPho19

[54] were streaked on 5-fluoro-orotic acid-containing medium to

generate HLY4032 and HLY4039 (Ura2 strains for hda1/hda1

and rpd3/rpd3 mutants). Ura+ strains of TS3.3 [85] and HLY4032

were obtained by transforming AscI digested pBES116 [37], which

were used for hyphal induction. hda1/hda1 yng2/yng2 mutants were

generated by deleting YNG2 in HLY4032 [50]. The disruption was

confirmed by southern blotting (unpublished data).

A 0.9-kb PCR product (primers 1 and 2) containing the C-terminal

NRG1 coding region and a 1.3-kb PCR product (primers 9 and 10)

containing the C-terminal RPD3 coding region were inserted into the

BamHI-MluI sites of pPR673 [50]. The resulting plasmids were

digested with SacI to target integration into their own loci to express

Nrg1-13Myc and Rpd3-13Myc. A 1.3-kb PCR product (primers 5

and 6) containing the C-terminal HDA1 coding region and a 0.8-kb

PCR product (primers 7 and 8) containing the C-terminal YNG2

coding region were digested with BglII and MluI and cloned into the

BamHI-MluI sites of pPR673. The resulting plasmids were digested

with BamHI to target the integration of the plasmid into its own locus

to express Hda1-13Myc and Yng2-13Myc. The pMAL2-NRG1-

13Myc plasmid was constructed by amplifying NRG1-13MYC

(primers 3 and 4) from pACT1-NRG1-13MYC plasmid (our

unpublished data). The PCR product was digested with XbaI and

KpnI to replace WOR1-HA from pMAL2-WOR1-HA plasmid [86].

The resulting plasmids were digested with AscI to target integration

into the ADE2 locus to express Nrg1-13Myc.

The YNG2 coding sequence was amplified using primers 13 and

14. The resulting 0.9-kb PCR product was digested with NotI and

MluI and inserted into the NotI-MluI site of pPR673 to create

pACT1-YNG2-13MYC. A 1.6-kb YNG2 promoter fragment

upstream of the START site of YNG2 (from 21 to 21,600) was

PCR amplified using primers 11 and 12. The resulting purified

PCR product was digested with StuI and BamHI and cloned into

the StuI-BamHI site of pACT1-YNG2-13MYC, displacing the 1-kb

ACT1 promoter region to generate pYNG2-YNG2-13MYC. Two-

step PCR was used to create pYNG2-YNG2K175R-13MYC. Two

pairs of primers (primers 13 and 15, 16 and 14) were used to PCR

amplify overlapping YNG2 fragments with the mutation in the

overlapping region. The resulting PCR products were purified and

mixed as templates for another round of PCR amplification using

the primers 13 and 14, which produced the full-length YNG2K175R

sequence. The resulting mutant, YNG2K175R, was cloned into the

NotI-MluI site of the plasmid pYNG2-YNG2-13MYC, replacing

the wild-type copy, and was confirmed by DNA sequencing. Two-

step PCR was used to create pYNG2-YNG2K175Q-13MYC. Two

pairs of primers (primers 13 and 17, and 18 and 14) were used to

PCR amplify overlapping YNG2 fragments with the mutation in

the overlapping region. Subsequent steps were done by using the

same methods as pYNG2-YNG2K175R-13MYC. These plasmids

were digested with the PmlI within the YNG2 promoter region for

integration into the endogenous YNG2 locus in yng2/yng2 and

hda1/hda1 yng2/yng2 mutant strains.

Northern and Quantitative RT-PCR Expression AnalysisMethods for RNA isolation and Northern blot hybridization

were carried out as previously described [87]. Probes for HWP1,

ALS3, ECE1, and ACT1 were made by PCR amplification of

,500-bp fragments from coding regions of each gene from

SC5314 genomic DNA. The primers used were as follows: HWP1,

primers 19 and 20; ALS3, primers 21 and 22; ECE1, primers 23

and 24; and ACT1, primers 25 and 26. For quantitative real-time

reverse transcription-PCR (qRT -PCR) analysis, 10 mg of total

RNA was DNase-treated at 37uC for 1 h using the DNA-free kit

(Qiagen), cDNA was synthesized using the SuperScript II Reverse

Transcriptase kit (Invitrogen), and qPCR was done using the iQ

SYBR Green Supermix (Bio-Rad) using the primers 27 and 28 for

HWP1, primers 29 and 30 for ALS3, primers 31 and 32 for ECE1,

primers 33 and 34 for ACT1, and primers 35 and 36 for NRG1.

The iCycler iQ detection system (Bio-Rad) was used with the

following program: initial denaturation at 94uC for 5 min,

followed by 40 cycles of 94uC for 20 s, 56uC for 30 s, and 68uCfor 20 s. Amplification specificity was determined by melting curve

analysis.

Chromatin ImmunoprecipitationChromatin immunoprecipitation was performed as described

with modifications [50,57]. Cells were formaldehyde cross-linked

by adding formaldehyde (37%) to a 1% final concentration.

Treated cultures were mixed by shaking and incubated for 15 min

at room temperature. 2.5 M glycine was added to a final

concentration of 125 mM, and treated cultures were mixed and

incubated for 5 min at room temperature. Cells were pelleted at

3,000 g for 5 min at 4uC, washed four times with 20 ml PBS, and

resuspended in 400 ml of 4uC lysis buffer (50 mM HEPES-KOH

[pH 7.5],140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1%

sodium deoxycholate) with protease inhibitors. All subsequent



ChIP and wash steps were done at 4uC. Cells were lysed using a

Fast-Prep system (FP120; Thermo Electron, Waltham, MA). DNA

was sheared by sonication six times for 20 s at high power on a

Bioruptor (diagenode) with 40 s intervals on ice.

For the IP, 10 ml of anti-Myc (SC-789, Santa Cruz), 4 ml of anti-

H3 (ab1791; Abcam) or anti-acetylated-H4 (06-866; Millipore)

antibodies were used for ,4 mg of chromatin proteins in an

immunoprecipitation volume of 200 ml. The IP was incubated O/

N at 4uC, with agitation. Then 50 ml of a 50% suspension of

protein A-Sepharose beads (GE Healthcare, 17-0974) in lysis

buffer was added to the IP and incubated 2 h at 4uC with

agitation. The beads were pelleted for 1 min at 3,000 g. After

removal of the supernatant, the beads were washed with a series of

buffers for 5 min each wash: twice in lysis buffer, twice in high-salt

lysis buffer (50 mM HEPES-KOH [pH 7.5], 500 mM NaCl,

1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate),

twice in wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl,

0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and

once in TE (10 mM Tris, 1 mM EDTA [pH 8.0]). After the last

wash, 75 ml of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM

EDTA, 1% SDS) was added to each sample, and the beads were

incubated at 65uC for 10 min. The beads were spun for 1 min at

5000 g, and the supernatant was removed and retained. A second

elution was carried out with 75 ml of elution buffer 2 (TE, 1%

SDS) and eluates from the two elution steps were combined. For

the ChIP input material set aside, 140 ml elution buffer 2 (TE, 1%

SDS) was added to 10 ml WCE. ChIP and input samples were

incubated overnight at 65uC to reverse the formaldehyde cross-

links. 150 ml of proteinase K solution (TE, 60 mg/ml glycogen,

500 mg/ml proteinase K) was added to each sample, and samples

were incubated at 37uC for 2 h. Samples were extracted twice with

400 ml Tris buffer-saturated phenol/chloroform/isoamyl alcohol

solution (25:24:1). 15 ml of 5 M NaCl and 0.8 ml of 100% ethanol

(4uC) were added and the DNA was precipitated for 20 min at

220uC, then pelleted by centrifugation at 14,000 g for 10 min at

4uC, washed once with cold 70% ethanol, and allowed to air dry.

The samples were resuspended in 30 ml of TE containing 100 mg/

ml RNaseA and incubated for 1 h at 37uC. DNA derived from the

whole cell extracts and immunoprecipitation (IP) eluate was

analyzed by quantitative PCR (qPCR). Plotted are the average

and standard error of the mean of three independent qPCR

reactions for each experiment.

ImmunoprecipitationAcetylated Yng2-Myc was immunoprecipitated with 30 ml

protein A-sepharose beads (GE Healthcare, 17-0974) conjugated

with 10 ml of rabbit polyclonal anti-acetylated-lysine (Cell

Signaling, 9441S) at 4uC overnight in 200 ml pre-cleared WCEs,

and detected with a peroxidase-conjugated anti-c-Myc antibody

(Roche).

Supporting Information

Figure S1 Nrg1 is detected in the nucleus of both apical and

subapical cells of hyphae. Wild type cells carrying Nrg1-Myc

(HLY3922) were processed for indirect immunofluorescence, as

described [88]. Cells were grown in YPD+10% Serum medium at

37uC for hyphal induction or in YPD medium for yeast growth.

Cells were fixed at 5 h after induction and stained for Nrg1-Myc

with 9E10 mouse antibodies (Covance) and FITC-conjugated

secondary antibodies (Jackson Laboratory). DNA was stained with

DAPI. An untagged control (SC5314) was included.

(TIF)

Figure S2 Promoter recruitment of Hda1 is required for

hyphal maintenance by inhibiting Nrg1 access to the promoters

of hypha-specific genes. (A) ALS3 and ECE1 mRNA levels were

determined by qRT-PCR as described in Figure 3 (B) Kinetics

of Nrg1-Myc (B) and Hda1-Myc (C) binding at the ALS3 and

ECE1 promoters were determined by ChIP as described in

Figure 3C,D.

(TIF)

Figure S3 Rpd3-Myc disassociates rapidly from the promoters

of hypha-specific genes upon hyphal induction, determined by

ChIP as described in Figure 1B.

(TIF)

Figure S4 The function of Hda1 in sustained hyphal transcrip-

tion is mediated through Yng2 deacetylation (A) ALS3 and ECE1

mRNA levels were determined by qRT-PCR as described in

Figure 4(B) Relative Yng2 enrichment (B), relative H3 occupancy

(C), and H4 acetylation level (D) at ALS3 and ECE1 promoter.

ChIPs were performed as described in Figure 4E,F,G.

(TIF)

Figure S5 Kinetics of Yng2-Myc and Yng2K175R-Myc promoter

binding in hda1 mutants by ChIP with anti-Myc as described in

Figure 5C.

(TIF)

Table S1 Yng2 deacetylation by Hda1 is not required for germ

tube formation. (A) Germ tube formation of wild type and hda1/

hda1. Cells of wild type (TS3.3+pBES116) and hda1/hda1

(HLY4032+pBES116) were diluted 1:100 into indicated medium

at 37uC. The percentage of cells forming germ tubes in YPD+10%

serum medium, Spider medium, and M199 pH 8 medium at

60 min or in Lee’s medium at 180 min was determined by

counting at least 300 cells/sample, in triplicate. The samples from

Spider medium were gently sonicated to disrupt clumping. Mean

(% germ tube formation) 6 SE (standard error) of two

independent experiments. The hda1/hda1 mutant is able to form

germ tube in YPD with serum and Spider media but shows a

dramatically reduced level of germ tube formation in M199 and

Lee’s media. This is likely due to impaired growth of the mutant in

the media. The doubling time of the wild type (TS3.3+pBES116)

and hda1/hda1 (HLY4032+pBES116) in YPD medium at 30uC is

105 min and 135 min, respectively, and in M199 PH 8 medium at

30uC is 150 min and over 18 h, respectively. The defect of hda1/

hda1 cells in germ tube formation in M199 is consistent with the

report by Zacchi et al. [89]. (B) yng2K175Q mutant has no dramatic

defect in germ tube formation. Cells of wild type YNG2

(HLY4035), yng2K175R (HLY4036), and yng2K175Q (HLY4037) were

diluted 1:100 into indicated medium at 37uC. The percentage of

cells forming germ tubes was calculated as described in (A). The

two yng2 mutants show a similar growth rate as the YNG2 strain in

all media examined.

(PDF)

Acknowledgments

We thank Drs. D. Soll, J.Y. Chen, M. Whiteway, A. Mitchell, and J. Ernst

for C. albicans strains, and G.R. Fink for comments on the manuscript.

Author Contributions

Conceived and designed the experiments: YL AW HL. Performed the

experiments: YL CS AW. Analyzed the data: YL CS HL. Wrote the paper:

YL HL.



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